This application gains priority from provisional application No. 60/139,625 filed Jun. 17, 1999 herein incorporated by reference.
Elastase is a general term that describes a group of protease enzymes that have the ability to degrade elastin. Elastin is the primary extracellular matrix protein that confers elastic qualities to a variety of tissues including the lung, skin and blood vessels. Different proteases from the serine, cysteine and metallo classes have been shown to degrade elastin with varying degrees of activity. In addition to elastin, serine elastases have been shown to degrade or process other proteins with varying relative activities. The serine elastases share the property of preferential cleavage of polypeptides and proteins adjacent to aliphatic amino acid residues, primarily alanine, valine and methionine. These enzymes also cleave, to a variable extent, at sites adjacent to leucine and isoleucine.
Examples of serine elastases include pancreatic elastase (PE), neutrophil elastase (NE), proteinase-3 (PR-3), endogenous vascular elastase (EVE), endothelial cell elastase (ECE)) and at least three other serine elastases including those derived from transformed rat liver epithelial, Schwann cells and human carcinoma cell lines, human skin fibroblasts and human lymphocytes.
Among elastases, the most well studied enzymes are PR-3 and NE. These enzymes are structurally similar but biologically different. Both Proteinase-3 (PR-3) and NE are co-localized in neutrophil primary granules and are co-released from activated human neutrophils. Both enzymes degrade elastin when purified enzyme and substrate are incubated together. However, despite structural similarities, not all endogenous inhibitors of NE inhibit PR-3, for example secretory leukocyte protease inhibitor (SLPI). Furthermore, the biology of NE and PR-3 appears to be significantly different.
NE appears to be primarily responsible for degradation of extracellular matrix (ECM) proteins and other important substrate proteins (immunoglobulins, surfactant apoproteins, etc.). Both NE and PR-3 play roles in the activation of pro-enzymes such as metalloproteinases (MMPs). In contrast, PR-3 rather than NE appears to be particularly well-suited to the processing of pro-cytokines to their active biological forms. The amount of at least two of the more important pro-inflammatory cytokines, produced by monocytic cells, TNF-xcex1 and IL-1xcex2 has been shown to be differentially enhanced by PR-3 relative to NE. It has also been shown that PR-3, but not NE, can process mature interleukin-8(77) having 77 amino acid residues to a form having approximately 10 fold greater biological activity interleukin-8 (70) having 70 residues.
Inflammatory cell serine elastases (and metalloproteinases) are critical enzymes for directed cell migration of both neutrophils and monocyte/macrophages. Their roles in this context were thought to be limited to the degradation of vascular basement membrane and underlying extracellular matrix proteins. In addition to their activities as matrix degrading enzymes, however, their ability to affect local regulation and amplification of the inflammatory response suggests a broader role in a variety of different disease states.
Some pathological conditions are believed to result at least in part from an imbalance between the elastases and their endogenous inhibitors. Uncontrolled proteolytic degradation by neutrophil elastases, especially NE has been implicated in a number of pathological conditions like pulmonary emphysema, acute respiratory distress syndrome, septic shock, multiple organ failure, rheumatoid arthritis, and cystic fibrosis.
One approach to disease management includes therapeutic intervention with small molecule elastase inhibitors for blocking the activity of particular elastases. For instance, U.S. Pat. No. 5,618,792 to Gyorkos et al., as well as continuation-in-part U.S. Pat. Nos. 5,807,829; 5,861,380; 5,869,455; 5,874,585; and 5,891,852 describe small molecule inhibitors and their method of synthesis that are selective for human neutrophil elastase (NE). These inhibitors of NE were shown to be effective in attenuating elastases-induced lung injury. These patents are hereby incorporated herein by reference.
There has been extensive efforts focused on NE inhibition in a variety of pathological conditions such as pulmonary emphysema, acute respiratory distress syndrome, septic shock, multiple organ failure, rheumatoid arthritis, and cystic fibrosis. This has not been the case for inhibitors of elastases other than neutrophil elastase. Such inhibitors would be useful for enhancing the treatment of the above stated pathologies. Furthermore, elastase inhibitors having specificity for elastases other than NE, such as PR-3, would be useful for treating pathologies such as vascular and inflammatory disorders including restenosis, atherosclerosis and vasculopathy, myocardial infarction, stroke and bronchopulmonary dysplasia.
Various embodiments of the present invention provide inhibitors and methods of inhibiting the activity of a plurality of serine elastases using a single inhibitor that may have specificity for elastases including neutrophil elastase or have specificity for elastases other than neutrophil elastase.
Accordingly, in a preferred embodiment of the invention, there is provided a serine elastase inhibitor that provides balanced inhibitory activity with respect to a plurality of serine elastases including an agent having a chemical structure including a serine elastase recognition moiety and a warhead moiety. More particularly, the agent may inhibit neutrophil elastase and PR-3. The agent may provide balanced inhibitory activity as a first inhibitory constant for NE and a second inhibitory constant for PR-3 that may differ by no more than two orders of magnitude, more specifically by fifty fold, and preferably by no more than one order of magnitude.
In another embodiment, one of the serine elastase recognition moiety (SERM) and the warhead moiety (WHM) contains a carbonyl group, the SERM further containing a first submoiety, the warhead further containing a heterocycle warhead submoiety, such that a carbonyl carbon of the carbonyl group directly attaches to a carbon of the heterocycle submoiety, in addition to the first submoiety. The SERM may contain one of a plurality of first submoieties. In a preferred embodiment, a method is provided that includes administering to an environment containing serine elastase, an effective amount of a serine elastase inhibitor, the agent having a chemical structure including a serine elastase recognition moiety and a warhead moiety, the agent being provided in a pharmaceutically acceptable carrier, the serine elastase inhibitor having balanced inhibitory activity with respect to a plurality of serine elastases. The serine elastases include neutrophil elastase and PR-3.
In a preferred embodiment of the invention, a compound is provided with the formula: 
wherein Z is selected from the group consisting of: any of the structures listed in FIG. 5 or any derivatives or analogs thereof.
R1 is selected from the group consisting of methyl (xe2x80x94CH3), ethyl (xe2x80x94CH2CH3), propyl (xe2x80x94CH2CH2CH3), butyl (xe2x80x94CH2CH2CH2CH3), (xe2x80x94CH2CH2xe2x80x94Sxe2x80x94CH3), (xe2x80x94CH2xe2x80x94Sxe2x80x94CH3), (xe2x80x94CH2CH2xe2x80x94Oxe2x80x94CH3), (xe2x80x94CH2xe2x80x94Oxe2x80x94CH3), and (xe2x80x94CH2xe2x80x94Oxe2x80x94CH2CH3)
corresponding to the side chains of alanine, aminobutyric acid, norvaline, norleucine, methionine and homomethionine.
R2 is selected from the group of phenyl, cyclohexyl, morpholino, H;
R3 is selected from the group of phenyl, cyclohexyl, morpholino, H;
R4 is H or, 
R5 is selected from alkyl, alkenyl, haloalkyl, haloalkenyl, alkynyl being linear or branched; a phenyl, phenylalkenyl, or phenylalkyl optionally substituted with halogen, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamido, arylcarboxamido, alkylthio, or haloalkylthio groups being linear or branched; a heteroaryl, heteroarylalkyl or heteroarylalkenyl wherein the heteroaryl group is a monocyclic five or six membered ring containing one or two heteroatoms independently selected from oxygen, sulfur and nitrogen optionally substituted with halogen, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, alkyl, alkoxy, alkenyl, alkynyl, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamido, arylcarboxamido, alkylthio or haloalkylthio groups being linear or branched; and X and Y are independently O, S or N wherein N is optionally substituted with alkyl, alkenyl, alkynyl being linear or branched; a phenyl, phenylalkenyl, or phenylalkyl optionally substituted with halogen, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, alkyl, alkenyl, alkynyl, alkoxy, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamido, arylcarboxamido, alkylthio, or haloalkylthio groups being linear or branched; a heteroaryl, heteroarylalkyl or heteroarylalkenyl wherein the heteroaryl group is a monocyclic five or six membered ring containing one or two heteroatoms independently selected from oxygen, sulfur and nitrogen optionally substituted with halogen, cyano, nitro, haloalkyl, amino, aminoalkyl, dialkylamino, alkyl, alkoxy, alkenyl, alkynyl, haloalkoxy, carboxyl, carboalkoxy, alkylcarboxamido, arylcarboxamido, alkylthio or haloalkylthio groups being linear or branched, provided at least one of X or Y is N; and provided that where both X and Y are N, only one of X or Y is substituted; R1, R2, R3, R4, R5 being further selected from any of the structures described in FIG. 3.
In a preferred embodiment, a pharmaceutical formulation is provided for treating an elastase induced pathology, that includes an effective dose of a compound such as described above.
In a further embodiment of the invention, a method is provided for inhibiting proteinase-3 or neutrophil elastase, or proteinase-3 and neutrophil elastase, in which a compound according to any listed above is administered in an effective dose in a pharmaceutically acceptable formulation.
Additional embodiments of the invention include providing balanced inhibitory activity for a plurality of elastases that further include endovascular (EVE), or endothelial cell (ECE).